Shigella is a Category B Priority Pathogen that causes disease by invading and spreading through the human colonic mucosa. We have shown that a mutation in the Shigella locus encoding two genes known to be important in virulence of enteric pathogens (virK and msbB2) leads to alterations in the levels of several membrane proteins. As MsbB2 is known to myristoylate lipid A of LPS, our results suggest that VirK and/or MsbB2 may affect not only LPS, but also protein composition of the membrane. One of the membrane proteins that is affected is IcsP, the outer membrane protease that proteolyzes the Shigella actin-based motility protein IcsA at the bacterial surface. Our preliminary data are consistent with a model in which VirK and MsbB2 are in the same biochemical pathway in terms of their effects on IcsP, suggesting they may be in the same pathway with respect to all affected membrane proteins. Our long-term goal is to understand the mechanisms by which VirK and MsbB2 modulate virulence of enteric pathogens. Given our long experience with IcsP and the availability in our laboratory of molecular tools for the study of IcsP, we are in a unique position to efficiently perform experiments in which IcsP is used as a reporter. Consistent with the exploratory nature of R21 applications, the goal of this R21 proposal is to use IcsP as a reporter in exploratory studies of the mechanism(s) by which VirK and MsbB2 affect Shigella virulence. Our Specific Aims are as follows: 1. Analysis of Shigella VirK and MsbB2 function, using IcsP expression as a reporter; and 2. Characterization of the mechanism of VirK- and/or MsbB2-dependent regulation of icsP transcription in response to pH. Results from these studies will serve as a springboard from which to characterize the relevance and breadth of this pathway in Shigella and in other enteric pathogens that contain VirK and/or MsbB2. These exploratory studies are highly likely to result in specific testable hypotheses that are relevant to disease. [unreadable] [unreadable] [unreadable]